Imagej software

Total protein was extracted from the left ventricular apex of mice using the Total Protein Extraction Kit (SD-001, Invent, USA). Protein lysates were extracted from cultured cells using RIPA lysis buffer (R0010, Solarbio, China) supplemented with a protease inhibitor cocktail (CWbio, China). Equal amounts of protein samples were separated on 10 to 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% skim milk for 1 hour at room temperature and incubated with primary antibodies at 4°C overnight. On the next day, the membranes were incubated with peroxidase-conjugated secondary antibodies (1:5000, Jackson ImmunoResearch Laboratories, USA) for 1 hour at room temperature, followed by detection using a chemiluminescent substrate (Millipore, USA) and chemiluminescent instrument (GE, Amersham Imager 680RGB, USA). Images were analyzed with ImageJ software (Media Cybernetics Inc., USA), and β-actin was used as a control to verify equal protein loading. The primary antibodies used were listed in table S1.

After the corresponding treatment, neurons grown on coverslips (1 × 10⁶ cells per well) were fixed with 4% paraformaldehyde for 30 min, washed with PBS, and then permeabilized with 0.1% Triton X‐100 (Sigma–Aldrich). After blocking with 5% bovine serum albumin, the coverslips were incubated at 4°C overnight with anti‐rabbit cleaved caspase‐3 antibody (1:150; Cell Signaling Technology). The coverslips were then rinsed and incubated with Alexa Fluor 594‐conjugated donkey anti‐rabbit IgG (1:200; Cell Signaling Technology) for 2 h. After thorough rinsing, the nuclei were stained with 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI; 1 μ/ml) and then observed by fluorescence microscopy (OLYMPUS BX53). The number of immunoreactive cells was quantified with ImageJ software (Media Cybernetics, Inc.). Negative controls were performed by omitting the application of the primary antibody. Five different fields for each sample were counted. We calculated the percentage of positive cells with cleaved caspase‐3 expression (red) with respect to the total number of DAPI (blue). All counts were conducted by a blinded investigator. Each experiment was replicated six times.

Total levels of ROS and superoxide were detected by using the intracellular ROS/superoxide detection assay kit (ab139476; Abcam). Neurons cultivated in coverslips at a density of 1 × 10⁶ cells per well (12‐well plates) after the corresponding treatment for 24 h were washed with fresh medium and loaded with the OS detection reagent (1:500) and superoxide detection reagent (1:500) for 30 min at 37°C. For the negative control, an ROS inhibitor (N‐acetyl‐l‐cysteine) at a final concentration of 10 mM was added to the cells of the OGD group for 30 min before the detection reagent was loaded. After the cells were washed three times with wash buffer, ROS‐ and superoxide‐positive cells were observed by fluorescence microscopy (OLYMPUS BX53) with the recommended filter set for ROS detection compatible with fluorescein (Ex/Em = 490/525 nm) and superoxide detection filter set compatible with rhodamine (Ex/Em = 550/620 nm). Increased levels of OS result in uniform green cytoplasmic staining in the presence of the OS detection reagent (green) and a bright orange nuclear staining pattern in the presence of the superoxide detection reagent (red). The mean fluorescence intensity was analyzed with ImageJ software (Media Cybernetics, Inc.). All samples were repeated six times.